Method for clotting heparized blood by the addition of protamine, thrombin and snake venom

ABSTRACT

A cocktail reagent preparation for the rapid production of serum contains thrombin, snake venom, and protamine sulfate. The preparation employs very small quantities of clot promoting substances which behave in a synergistic manner such that rapid clotting of highly heparinized blood is achieved without altering the chemical analysis of the blood enzymes, proteins, sugars, or electrolytes. Thus, clinicians who rely upon the results of such tests can more closely monitor organ and tissue function and adjust patient therapies accordingly.

FIELD OF THE INVENTION

The present invention relates, in general, to the production of serum,and, more particularly to a reagent preparation for the rapid productionof a high quality serum specimen upon which diagnostic blood chemicalanalyses can be performed.

BACKGROUND OF THE INVENTION

Serum represents the liquid portion of the blood which remains followingthe clotting of the formed blood elements and blood coagulation factors.The serum contains various blood proteins, sugars, fats, enzymes andcharged particles (i.e., electrolytes) which are necessary for normalmetabolic processes. Devoid from the serum are the elements consumedduring the clotting process namely red blood cells, white blood cells,platelets and the blood coagulation factors. Serum, obtained bycentrifugation of clotted blood, is used to conduct chemical analyseswhich are valuable tests used to diagnose and monitor muscle and organfunction, metabolic balances, and basic physiologic functions. Byremoving the substances consumed during clotting, a better substrateupon which to perform chemical analyses is produced. Thus, serum is thepreferential test substrate on which to perform analytical tests such asenzyme, electrolyte, protein and glucose assays since the interferenceof unwanted substances has been removed. These analytical tests areroutinely performed using automated chemistry analyzers such as theHitachi 717, Technicon SMAC, and Ciba-Corning 5500 Express.

In the past, the production of serum from whole blood has been a passiveprocess in which freshly collected blood is added to a glass test tubeand allowed to clot. Blood, once removed from the body, has a naturaltendency to clot and its exposure to a surface such as glass promotesclotting in a more efficient manner. Contact with a glass surface causesthe activation of coagulation factors which interact in a mechanismcommonly referred to as the coagulation cascade. In this process, aninactive coagulation factor is chemically converted to an active enzymewhich subsequently converts yet another inactive precursor. Thecoagulation factors are identified by Roman Numerals; Factors I-XIII.The end result of the coagulation cascade is a conversion of the solubleplasma protein fibrinogen, to an insoluble protein, fibrin, whereby thefibrin clot entraps the white cells, red cells, and platelets forming asolid gelatinous mass. Substances not consumed in the process remainfree of the gelatinous mass and are found in the liquid matrix. It isthis liquid portion which serves as the best test substrate upon whichto perform serum chemistry analyses.

The passive clotting process described above has several inadequacies.Blood from normal healthy individuals will routinely clot inapproximately 6 to 10 minutes in a glass test tube. However, blood fromsick individuals who may have deficiencies of coagulation proteins orfrom patients who are receiving anticoagulation therapy (i.e., oralanticoagulants or heparin) may require extensive time to clot (i.e., 2-8hours). Typically highly heparinized blood is between 2-5 units permilliliter and possibly higher. Consequently, in the past, there hasbeen a delay associated with the obtaining of blood specimens and theperformance of the analytical tests, thereby affecting the ability ofthe clinician to quickly provide optimal patient care. In addition, theblood from individuals with deficiencies of coagulation proteins orpatients receiving anticoagulation therapy may never form a complete andadequate fibrin mass. Incomplete clotting in heparinized blood specimensresults in a poor quality serum substrate upon which to perform thechemical test. Furthermore, heparin anticoagulated blood which ma notclot initially may actually begin to clot once placed in the chemistryanalyzer, thereby clogging the system and causing an instrumentshutdown.

In order to improve the clot forming process, laboratorians haveroutinely added the clot promoting agent thrombin to the blood specimen.Thrombin actively converts fibrinogen to fibrin, thereby forming theclot more efficiently then the slower glass-activated clotting process.Although thrombin may improve clotting in normal blood, its effects onheparinized blood are minimal since heparin acts as an inhibitor ofthrombin. As a result, collection of heparinized blood in these thrombincontaining serum test tubes provides little improvement of the serumspecimens, both in quality as well as in the time required to preparethe specimens, as compared to the plain glass test tube. Thrombincontaining test tubes are available from Becton-Dickinson and Company,VACUTAINER Systems, of Rutherford, N.J. 07070. Though it is possible toovercome the anticoagulant effect of heparin with very large amounts ofthrombin, the fibrin mass so formed is often an incomplete one.Consequently, following the centrifugation of an apparently clottedspecimen, delayed clotting occurs in the serum supernatant rendering itof little use in analytical tests. Moreover, such an excessive thrombinquantity will interfere with the proper performance of the chemicalanalyses, thereby altering the test results. Furthermore, excessiveprocoagulants such as thrombin can cause some red blood cell hemolysis,whereby the red blood cell bursts open contaminating the serum specimenwith intracellular particles. The above stated problems relating to therapid production of a high quality serum specimen from heparinizedpatients on which to perform chemical analyses are significant sincemany patients receiving heparin anticoagulation therapy requirerepetitive serum chemistry analyses.

SUMMARY OF THE INVENTION

The problems and disadvantages associated with the prior art methods ofobtaining serum discussed above are overcome in accordance with thepresent invention by providing a cocktail preparation to enablerelatively rapid clotting of highly heparinized blood when thepreparation is mixed with a given volume of blood. The preparationcontains thrombin, a heparin neutralizing substance, and a snake venomwhich is capable of converting fibrinogen to fibrin and is unaffected bythe presence of heparin. The thrombin, heparin neutralizing substance,and snake venom are present in the preparation in sufficient amounts torapidly clot the highly heparinized blood sample to enable the serum tobe separated from the clotted matter. The serum is then chemicallytested to provide clinically accurate blood chemistry results.

By providing such a clot promoting cocktail, it is now possible toprepare serum specimens and obtain accurate blood chemistry analyses ofhighly heparinized patients in a rapid fashion. Clinicians who rely uponresults of such tests to monitor and adjust patient therapies now havethe means to more closely identify patient deficiencies and provide theappropriate treatment. Thus, the patient benefits from a more closelymonitored therapeutic regimen.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The present invention describes a unique combination of clot promotingreagents which when combined with fresh normal blood, fresh blood fromcoagulation deficient patients, or fresh blood from heparinized patientsresults in rapid clotting of the blood. By applying this reagentcombination in very low concentration it is possible to clot nonanticoagulated blood from normal individuals within two minutes andblood from patients receiving heparin therapy within five minutes. Priorto the development of this clot promoting preparation, nonanticoagulated blood required three to five times as long to clot (i.e.,6-10 minutes) and heparinized blood would not clot for several hours.Some heparinized blood samples actually remained non-coagulated after6-8 hours.

The reagents used to formulate the cocktail include two clot promotingenzymes, thrombin and snake venom, and a heparin neutralizing reagent,protamine sulfate. Thrombin may be obtained from the plasma of severalanimal species including, but not limited to, cows (i.e., bovine),sheep, horse, pig, rat, mouse, and humans. In the preferred embodiment,thrombin from a bovine source is used. The snake venoms may be derivedfrom one of several species of snakes which convert fibrinogen to fibrinand are unaffected by the presence of heparin, including, but notlimited to the family of snakes known as the Genus: Agkistrodon,Bothrops, Crotalus, Eschis, and Trimeresurus. In the preferredembodiment, the venom from the snake Bothrops atrox, or alternately, thepurified procoagulant portion of the crude venom, atroxin, is used. Itis understood that the clot promoting effect requires the conversion offibrinogen to fibrin while being unaffected by heparin. Hence, it ispossible that recombinant forms of snake venom might be employed. Theprotamine sulfate is extracted from fish of the species salmon.Protamine sulfate is preferred, but protamine chloride (salmon) can beused as well. Essentially any heparin neutralizing preparation can beemployed such as polybrene, heparinase, and anion exchange resins suchas TEAE cellulose, DEAE cellulose providing that the blood chemistry isnot significantly altered.

The reagent preparation is prepared by combining individual reagentsinto a common batch. Appropriate stabilizers and buffers are added toinsure an acceptable shelf life. The combination of reagents may be usedin liquid form or alternatively may be freeze-dried using the basicprincipals of lyophilization. The liquid or dried powered reagent maythen be added directly to a blood collection test tube, oralternatively, to a special centrifugation container. Either glass orplastic containers may be used.

To prepare the reagent cocktail, individual stock reagents areassembled, rehydrated if required, and allowed to stabilize. In a firstcontainer, bovine thrombin is dissolved in barbital buffered saline(i.e., hereinafter "BBS"), to a final concentration of 100 USP units permilliliter (equivalent to approximately 1 mg/ml); the BBS iscommercially available from Sigma Chemical Co. St. Louis, Missouri.Bovine thrombin, commercially available from many sources, is easy toprepare and relatively inexpensive. Crude snake venom extract from thesnake Bothrops atrox is dissolved in BBS to a concentration of 1 mg/mlin a second container. Then, in a third container, protamine sulfate(from salmon) is dissolved in a sodium chloride (NaCl) solution to aconcentration of 10 mg/ml. A preservative such as thimerosal or sodiumazide may also be added at a final concentration of 0.02% to eachindividual container. The reagents are combined into a cocktailpreparation such that the final concentration in blood is 0.5 units ofthrombin per ml, 0.01 mg of snake venom per ml and 0.05 mg of protamineper ml. The reagents are in sufficient concentration such that a smallamount of this "cocktail" reagent is required to clot a blood specimen.This eliminates the possibility of a dilution effect creating falsechemistry analyses. If the addition of the cocktail results in adilution of the original blood volume by about 5% or more, this mayrender the final serum sample clinically unacceptable.

If the volume of blood to be clotted is 3 ml, a total of 0.04 ml of thecocktail reagent is prepared consisting of 0.015 ml of the thrombinsolution from the first container, 0.01 ml of the snake venom solutionfrom the second container, and 0.015 ml of the protamine sulfatesolution from the third container. Stock reagent preparations of greateror lesser concentration may also be employed and final quantities in thecocktail adjusted to achieve the desired preferred concentration. Thevolume 3 ml was selected as an example due to the fact that compactcentrifuges employ 3 ml as a typical load. Alternatively, we may collecta 6 ml blood sample by adding 0.08 ml of the reagent cocktail to theblood or a 9 ml blood sample by adding 0.120 ml of the cocktail, and soon. Preferred and acceptable formulations are as follows:

    ______________________________________                                        Formulation of the Reagent Preparation                                                                    Range of                                          Component  Preferred Concentration*                                                                       acceptable conc.                                  Reagent    (per milliliter) (per millimeter)                                  ______________________________________                                        Thrombin   0.5        units     0.2-3.0 units                                 Snake Venom                                                                              0.0033     mg        0.002-0.20                                                                            mg                                    Protamine Sulfate                                                                        0.05       mg        0.02-0.08                                                                             mg                                    BBS comprising                                                                sodium barbital                                                                          1.17       mg        0.39-2.34                                                                             mg                                    sodium chloride                                                                          1.46       mg        0.48-2.92                                                                             mg                                    sodium azide**                                                                           0.5        mg        0-1.0   mg                                    thimerosal**                                                                             0.2        mg        0-0.5   mg                                    ______________________________________                                         *The preferred concentration is the one used in the current cocktail          configuration and is the concentration of each component reagent as found     in the blood sample. These concentrations remain constant irrespective of     blood volume.                                                                 **Either sodium azide or thimerosal may be used as a preservative or          another appropriate antibacterial agent used.                            

As indicated, the cocktail as described above may be in liquid form.This liquid may then be placed in a collection reservoir to which bloodis subsequently added. Alternatively, the liquid cocktail may be placedin a collection reservoir, and the reservoir, either plastic or glass,may be freeze dried using conventional methods. A suitable freeze dryingapparatus (i.e., a lyophilizer) is sold by the Virtus Company ofGardineer, N.Y. and sold under the designation of Unitop 600. Thelyophilizer removes the water molecules such that the various proteinsand reagents remain in a dried (powdered) state. The freeze driedpreparation may be stored in any suitable container and subsequentlytransferred to an appropriate blood collection receiver. In a preferredembodiment, the entire solution is freeze dried to the inside of thecollection receiver. The collection reservoir may be one of severalconfigurations depending upon the apparatus to be used forcentrifugation, and for collection of the serum. The following examplesillustrate suitable collection reservoirs and centrifugation systems ofthe reagent cocktail use.

EXAMPLE 1

The reagents as combined to form the cocktail preparation are added to aglass or plastic test tube or collection reservoir. The amount of thecocktail preparation added is a function of the size of the reservoir asindicated above (i.e., 0.04 ml for 3 ml of anticipated sample). The tubemay be evacuated to a pre-determined amount and stoppered. The tube alsoserves as the blood collection reservoir as blood is added directly tothe tube, mixed, and allowed to clot. The clotted specimen is thencentrifuged in an appropriate centrifuge instrument and the serumremoved for testing.

EXAMPLE 2

According to Example 1 above wherein the cocktail preparation islyophilized in the test tube prior to evacuation and stoppering.

EXAMPLE 3

The cocktail preparation is placed in a small glass or plastic containerand stoppered. To use, the preparation is transferred to any bloodcollection reservoir and blood is subsequently added. Alternatively, thecocktail preparation may be added to a container of previously collectedblood.

EXAMPLE 4

As per Example 3 above wherein the cocktail preparation is lyophilizedin the container. Prior to use, the preparation is rehydrated withdistilled water in a volume equivalent to the original volume of thecocktail added.

When a patient's blood is added to the cocktail preparation, clottingrapidly occurs as a result of the following simultaneous reactions:First, thrombin immediately converts fibrinogen to fibrin, therebyforming the initial fibrin strands. Secondly, protamine sulfateneutralizes the anticoagulant effects of heparin if present in theblood. If heparin is absent from the blood, the protamine sulfate is ina sufficiently low concentration such that it does not affect the normalclotting process. Third, the procoagulant portion of the snake venomextract activates and cleaves the remaining fibrinogen molecules whichare ineffectively activated by the thrombin initially added. Throughthis enzymatic cleavage, all remaining fibrinogen is converted to fibrinresulting in the formation of a solid gelatinous mass consisting of theconsumed coagulation factors and the entrapped formed blood elements(i.e., the red blood cells, white blood cells and platelets).

The clotted sample may then be transferred to an appropriatecentrifugation container and spun at a high speed such that thegelatinous mass is forced to the bottom of the collection reservoir ortest tube and the liquid serum remains as a supernatant. This serumsupernatant may then be withdrawn from the collection reservoir using anappropriate pipetting device and transferred to the analytical cell ofthe chemistry analyzer. Any chemistry test normally performed on a serumpreparation may be performed on the serum preparation generated in thepresence of the clot promoting cocktail since the combination ofreagents employed in the cocktail does not alter the test results. Therapid availability of the serum preparation enables the clinician toformulate an appropriate clinical course of patient management byallowing the clinician to properly monitor organ and tissue function andto assess metabolic processes important to the health of the patient.

The advantages of this clot promoting cocktail are: the ability tocompletely clot highly heparinized blood in a very rapid manner (i.e.,less than 5 minutes), the ability to generate a high quality serumpreparation from which chemical analyses may be performed, and theunique use of synergistic effects of very low concentrations of reagentsin the preparation such that they do not interfere with the chemistryanalysis performed.

The most commonly used chemistry profile includes analysis of a group ofserum chemistry tests. This profile includes: electrolytes such assodium, potassium, chloride, and calcium; serum enzymes such as glutamicoxaloacetic transaminase (SGOT), gamma glutamyl transpeptidase (GGTP),lactic dehydrogenase (LDH), alkaline phosphatase (ALK), and creatinephosphokinase (CPK); and dissolved substances such as bilirubin,glucose, cholesterol, blood urea nitrogen (BUN), creatine, uric acid,albumin, and total protein.

In the example that follows, one aliquot of blood was allowed to clotpassively in a glass test tube (control) and a second aliquot wasclotted in a glass test tube using the clot promoting reagent cocktail(cocktail). In additional samples, heparin was also added before theclotting process was initiated. The chemistry profiles were obtained forall specimens. As shown below, the cocktail preparation did notstatistically alter (t-test of matched means) the chemistry testresults, which is of paramount importance. Furthermore, patients withabnormal blood chemistries (i.e., glucose or cholesterol levels) areaccurately measured using the cocktail preparation.

    ______________________________________                                        I. Non-heparinized blood                                                      TEST          COCKTAIL   CONTROL                                              ______________________________________                                        sodium        143.8 ± 4.3                                                                           141.0 ± 6.1                                       potassium     4.1 ± 0.3                                                                             4.0 ± 0.3                                         chloride      107.4 ± 5.0                                                                           106.8 ± 6.3                                       calcium       9.3 ± 0.4                                                                             9.0 ± 0.6                                         SGOT          20.0 ± 5.0                                                                            21.3 ± 5.6                                        GGPT          20.0 ± 10.9                                                                           20.4 ± 10.7                                       LDH           145.4 ± 34.2                                                                          164.8 ± 38.6                                      ALK           79.1 ± 18.5                                                                           76.0 ± 18.2                                       CPK           114.0 ± 62.8                                                                          109.2 ± 60.3                                      bilirubin     0.65 ± 0.38                                                                           0.60 ± 0.31                                       glucose       87.2 ± 7.2                                                                            83.2 ± 10.2                                       cholesterol   184.0 ± 25.5                                                                          178.0 ± 26.7                                      BUN           13.5 ± 4.0                                                                            13.4 ± 3.9                                        creatine      1.06 ± 0.2                                                                            1.06 ± 0.2                                        uric acid     4.6 ± 1.1                                                                             4.6 ± 1.4                                         albumin       4.7 ± 0.3                                                                             4.7 ± 0.4                                         total protein 7.0 ± 0.6                                                                             6.8 ± 0.6                                         ______________________________________                                    

    ______________________________________                                        II. Heparinized blood                                                         TEST          COCKTAIL   CONTROL                                              ______________________________________                                        sodium        143.7 ± 4.0                                                                           141.9 ± 7.0                                       potassium     3.6 ± 0.3                                                                             3.5 ± 0.3                                         chloride      110.0 ± 5.0                                                                           109.1 ± 6.8                                       calcium       8.3 ± 0.5                                                                             8.1 ± 0.7                                         SGOT          17.7 ± 4.2                                                                            16.9 ± 4.1                                        GGPT          19.9 ± 10.8                                                                           16.4 ± 9.1                                        LDH           135.9 ± 24.7                                                                          129.4 ± 24.8                                      ALK           73.2 ± 18.1                                                                           70.7 ± 14.6                                       CPK           87.0 ± 50.0                                                                           85.6 ± 48.0                                       bilirubin     0.63 ± 0.34                                                                           0.57 ± 0.30                                       glucose       83.8 ± 7.8                                                                            79.8 ± 10.9                                       cholesterol   161.1 ± 14.2                                                                          156.9 ± 21.9                                      BUN           12.1 ± 3.6                                                                            11.9 ± 3.7                                        creatine      0.9 ± 0.2                                                                             1.0 ± 0.2                                         uric acid     4.1 ± 0.8                                                                             4.1 ± 0.8                                         albumin       4.3 ± 0.3                                                                             4.2 ± 0.5                                         total protein 6.4 ± 0.6                                                                             6.2 ± 0.6                                         ______________________________________                                    

It will be understood that the embodiment described herein is merelyexemplary and that a person skilled in the art may make many variationsand modifications without departing from the spirit and scope of theinvention. All such modifications are intended to be included within thescope of the invention as defined in the appended claims.

I claim:
 1. A method for the rapid production of serum from a highlyheparinized patient, said serum to be chemically tested to provide bloodchemistry results, comprising:collecting a blood sample from said highlyheparinized patient; mixing a sufficiently small quantity of a clotpromoting cocktail with said blood sample such that said cocktail doesnot alter said blood chemistry results, said cocktail comprisingthrombin in a concentration range of about 0.2-3.0 units per milliliter,a protamine salt in a concentration range of about 0.02-0.08 milligramsper milliliter and a snake venom in a concentration range of about0.005-0.2 milligrams per milliliter which snake venom is capable ofconverting fibrinogen to fibrin and is unaffected by the presence ofheparin; waiting approximately five minutes during which time said bloodsample clots; centrifuging said clotted blood sample to form a serumsupernatant; and isolating said serum supernatant for blood chemicaltesting.
 2. The method according to claim 1, wherein said snake venom isselected from the family of snakes known as the Genus: Agkistroden,Bothrops, Crotalus, Echis, and Trimeresurus.
 3. The method according toclaim 2, wherein said snake venom is obtained from the snake Bothropsatrox.
 4. The method according to claim 1, wherein thrombin is obtainedfrom a bovine source.
 5. The method according to claim 1, wherein saidmixture contains 0.5 units of thrombin per milliliter, 0.01 milligramsof snake venom per milliliter, and 0.05 milligrams of protamine sulfateper milliliter.